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Abstract

The tropical earthworm Pontoscolex corethrurus (Rhinodrilidae, Oligochaeta) presents a broad distribution (e.g., 56 countries from four continents). It is generally assumed that temperature appears to limit the success of tropical exotic species in temperate climates. However, the distribution range of this species could advance towards higher elevations (with lower temperatures) where no tropical species currently occur. The aim of this study was to evaluate the soil and climatic variables that could be closely associated with the distribution of P. corethrurus in four sites along an altitudinal gradient in central Veracruz, Mexico. We predicted that the distribution of P. corethrurus would be more related to climate variables than edaphic parameters. Five sampling points (in the grassland) were established at each of four sites along an altitudinal gradient: Laguna Verde (LV), La Concepción (LC), Naolinco (NA) and Acatlán (AC) at 11–55, 992–1,025, 1,550–1,619 y 1,772–1,800 masl, respectively. The climate ranged from tropical to temperate along the altitudinal gradient. Ten earthworm species (5 Neotropical, 4 Palearctic and 1 Nearctic) were found along the gradient, belonging to three families (Rhinodrilidae, Megascolecide and Lumbricidae). Soil properties showed a significant association (positive for Ngrass, pH, permanent wilting point, organic matter and P; and negative for Total N, K and water-holding capacity) with the abundance of the earthworm community. Also there seems to be a relationship between climate and earthworm distribution along the altitudinal gradient. P. corethrurus was recorded at tropical (LV and LC) and temperate sites (NA) along the altitudinal gradient. Our results reveal that soil fertility determines the abundance of earthworms and site (climate) can act as a barrier to their migration. Further research is needed to determine the genetic structure and lineages of P. corethrurus along altitudinal gradients.

Introduction

Within soil biodiversity, earthworms are key components of the guild of ecosystem engineers (Jones, Lawton & Shachak, 1994). They provide a considerable level of ecosystem services, such as contributing to biogeochemical cycling and crop productivity (Turbé et al., 2010; Orgiazzi et al., 2016). Depending on their ecological classification (epigeic, endogeic or anecic), they can also modify the distribution and abundance of soil biodiversity, mainly by constructing structures and galleries within the soil profile and by producing casts and mucus (Turbé et al., 2010; Orgiazzi et al., 2016).

At the global level, apart from studies in North America where non-native earthworms are causing changes in soil biota and plant communities, little recognition has been given to invasions of soil organisms (Gates, 1954; Bohlen et al., 2004; Fahey et al., 2013). Among the 3,700 earthworm species described, approximately 3% (100–120) have been identified as invasive; for example, the ubiquitous Pontoscolex corethrurus (Müller, 1857), several temperate species from genera Amynthas and at least 10 species of Lumbricid (Brown et al., 2006; Beddard, 1912; Hendrix et al., 2008; Dupont et al., 2012). These earthworms have reached a broad distribution in many tropical and temperate agroecosystems and natural ecosystems. However, this has been facilitated by fishing bait, horticulture, waste management industries, road networks and vehicle transport which have contributed to surmount important biogeographic barriers (Eisen, 1900; Beddard, 1912; Gates, 1954; Hendrix et al., 2008).

In Mexico, since the early twentieth century, P. corethrurus has been the endogeic earthworm most commonly found in human-altered tropical ecosystems (Eisen, 1900; Lavelle et al., 1987; Brown et al., 2004; Fragoso & Rojas, 2014). However, the edge of the earthworm’s distribution range could advance towards higher elevations where few or no tropical species currently occur (Eisen, 1900; Beddard, 1912; Hendrix et al., 2008). The aim of this study was to evaluate the soil and climatic variables that could be closely associated with the distribution of P. corethrurus. We predicted that the distribution of P. corethrurus would be more related to climatic variables than edaphic parameters. We tested this hypothesis through a study of the earthworm community along an altitudinal gradient in central Veracruz, Mexico. In addition, we compared the occurrence of four possible situations in the altitudinal gradient (Marichal et al., 2010): (a) presence of P. corethrurus only, (b) coexistence of P. corethrurus and other species (native and invasive), (c) absence of P. corethrurus but presence of other species (native and exotic), and (d) absence of earthworms.

Methods

Study area

An altitudinal transect, ranging from 11 to 1,800 masl, was established in the central region of the State of Veracruz, Mexico. Five sampling points were established at each of four sites along this altitudinal gradient (Fig. 1): Laguna Verde (LV), La Concepción (LC), Naolinco (NA) and Acatlán (AC) at 11–55, 992–1,025, 1,550–1,619 and 1,772–1,800 masl, respectively.

Figure 1: Sampling sites of earthworms along an altitudinal gradient in central Veracruz, Mexico. For each site, the geographical coordinates (14N zone, Datum WGS84) are presented.

Climate information from the Mexican National Water Commission weather stations (http://www.conagua.gob.mx) was compiled for each site along the altitudinal transect. The monthly and annual 30-year averages were obtained for the following climate variables: average temperature (AT), average maximum temperature (AMT), average minimum temperature (AmT), total annual precipitation (TAP) and total evaporation (TE). With these data, climate types along the altitudinal gradient were determined using the Clima2 software (http://www.pablo-leautaud.com/home/proyectos/python/clima) and classified into one climate type according to the Köppen-Geiger system (Kottek et al., 2006; Peel, Finlayson & McMahon, 2007).

Earthworm sampling

For determining the distribution of earthworms, we choose the grasslands because they are a suitable habitat that fosters the growth of earthworms, and can be found along the altitudinal gradient. These grasslands are used as pasture for both an extensive (LV and LC) and semi-intensive cattle farming (NA and AC) under a rotational use of pasture (30–45 day grazing-rest cycle) and without the application of mineral fertilization (Lavelle, Maury & Serrano, 1981; Brown et al., 2004). The grasses species that grow in these grassland are: (a) native: sour paspalum Paspalum conjugatum P.J. Bergius (80% LV, 60% LC, 40% NA, and 40% AC), (b) introduced: bermudagrass or stargrass Cynodon nlemfuensis Vanderyst (20% LV, 40% LC, and 40% NA) and kikuyu Pennisetum clandestinum Hochst. Ex Chiov. (20% NA, and 60% AC). In the extensive production system (Dual-purpose cattle systems: milk and meat), cattle (Bos indicus × Bos Taurus cows: Zebu × Swiss or Holstein) was fed only on forage produced in grassland, ocassionaly suplemented with mineral salts. By contrast, under the semi-intensive system (milk), besides feeding on grassland forage, milk cows (Holstein) are given a dietary supplement made of carbohydrates (corn and barley stubble), protein (cane molasses, urea, dehydrated alfalfa and others) and mineral salts.

The quantitative sampling of earthworms was conducted along the altitudinal gradient (International Organization for Standarization, 2011). One monolith (25 × 25 × 30 cm deep) according to Anderson & Ingram (1993) was sampled at each of the five sampling points (in the grassland) established at least 200 m apart (Marichal et al., 2010), located at each of the four sites on the altitudinal transect, for a total of 20 monoliths along the altitudinal transect. Each monolith was separated into four strata: above-ground plant biomass, 0–10, 10–20 and 20–30 cm. Earthworms were then manually removed from each layer and preserved in 70% ethanol. In the laboratory, all specimens were fixed in 4% formaldehyde and then identified (to species or morphospecies), quantified and weighed. The sampling was conducted at the end of the rainy season (August–October 2011). Abundance and biomass data of the earthworms were converted into densities per square metre (ind. m−2 and g m−2, respectively) for each site (International Organization for Standarization, 2011).

Soil and foliage sampling

Prior to removing the earthworms, the biomass of grass was harvested from each monolith. In the laboratory, this plant material was dried (60° C for 72 h) and weighed, and its total nitrogen content (Ngrass) was determined using the Kjeldahl methods described in the Mexican Official Standard NOM-02-RECNAT-2000 (SEMARNAT, 2002).

Following the removal of earthworms, a 1-kg soil sample was taken from each stratum of each monolith. Soil samples were air-dried to constant weight and sieved (5 mm) to determine the texture (clay, silt and sand), water-holding capacity, permanent wilting point, pH, organic matter, Total C, Total N, P and K, using the methods described in the Mexican Official Standard NOM-02-RECNAT-2000 (SEMARNAT, 2002).

Statistical analysis

A one-way ANOVA was used to test for significant differences (P < 0.05) in soil properties between sites, using the Statistica software, ver. 7 (StatSoft, Tulsa, OK, USA).

We used a Linear Model (LM) to study earthworm abundance (ind. m−2) using soil properties and climatic elements. The effect of the climatic elements was included in the model through the variable “site” because it was possible to clearly distinguish the sites in a scatter plot (figure not shown) of the scores for the first two principal components obtained from a PCA of the climatic variables. The dependent variable (earthworm abundance) was transformed using natural logarithms because the empirical distribution of earthworms was highly asymmetric. All the analyses were performed using R software (R Core Development Team, 2015). We also fitted Linear Mixed Models to take into account the sampling design consisting in clustered samples and the response variable measured at two different scales, i.e., soil properties at the sample scale and climatic conditions at the site scale. The results showed that the variance component associated with the site effect was misleading. Apart from the linear models and before log-transforming the data, we also fitted generalized linear models with different families (e.g., Poisson, Negative Binomial); however, some convergence issues arose when fitting the models. Consequently, only the results for the fitted Linear Model are reported here.

Results

Site climate

The climate along the altitudinal gradient, from the lowest to the highest elevation sites, according to Kottek et al. (2006) and Peel, Finlayson & McMahon (2007) ranged from warm to humid tropical (Aw) to temperate (Cfb) (Table 1). There was a difference of approximately 11°C in the average, minimum and maximum temperatures between the lowest (LV, 11–55 masl) and the highest elevation sites (AC, 1,772–1,800 masl) along the gradient. Rainfall was higher in site LC (1676.8 mm) than in site LV (1143.0 mm), whereas sites NA and AC (1,550–1,619 and 1,772–1,800 masl) had intermediate values of 1,461 mm (Table 1).

Table 1:

Climate variables at the four sampling sites along an altitudinal gradient in central Veracruz, Mexico.

Soil properties and foliage

The physical and chemical variables of soil and nutritional quality of pasture along the altitudinal gradient displayed significant variations between tropical (LV and LC) and temperate (NA and AC) sites (Table 2). According to and the official Mexican standard soil fertility (SEMARNAT, 2002), soils from tropical sites had a heavy texture (clay loam; Regosols, Phaeozems and Vertisols; (Krasilnikov et al., 2013); were mildly acidic; and displayed intermediate values for water-holding capacity and permanent wilting point; were very rich in organic matter, total N and P; and were extremely poor in K. In contrast, soils from temperate sites had a light texture (loam; Andosols); greater water-holding capacity and permanent wilting point; were slightly acidic; and were very rich in organic matter, total N and P; and were extremely poor in K. Quality grass (Ngrass) in the temperate sites (NA and AC) was higher compared to tropical sites (LC an LC).

Table 2:

Soil properties and grass at the four sites along an elevation gradient in central Veracruz, Mexico.

Earthworm communities

Ten earthworm species (Annelida: Oligochaeta: Crassiclitellata) were found in the whole sampling (Table 3). Seven of these are well-known, ubiquitous species, some of which are considered invasive, belonging to three different families (Rhinodrilidae, Megascolecidae and Lumbricidae). The remainder of the earthworms were native morphospecies (differentiated from others only by morphological features). The highest diversity was found at site AC, with five species. The total abundance of the earthworm community ranged from 0 to 864 ind. m−2 (Fig. 2), with an average of 332 ind. m−2.

Table 3:

Earthworm species recorded in four sampling sites along an altitudinal gradient in central Veracruz, Mexico.

Figure 2: Abundance (A) and proportion (B) of Pontoscolex corethrurus and total earthworm community abundance (C) along an altitudinal gradient.

The LM analysis showed that the total abundance of the earthworm was significantly influenced by water-holding capacity (P = 0.026), permanent wilting point (P = 0.019), pH (P = 0.045), organic matter (P = 0.029), Total N (P = 0.015), P (P = 0.031), K (P = 0.016) and Ngrass (P = 0.009), while the climatic factors (sites) had no such effect (F = 5.57; P = 0.0652). That is, positive coefficients were associated with an increase in the number of earthworms, and negative coefficients were associated with a decrease in the number of earthworms (Table 4).

Table 4:

Estimated regression coefficients in the linear model that predict total earthworm abundance along an altitudinal gradient in central Veracruz, Mexico.

*The “site” was included in the linear model using Dummy variables with the reference cell method. The reference site was LV. We performed an analysis of variance for this linear model; the P-value associated to the site effect was 0.0652.

Pontoscolex corethrurus

Populations of P. corethrurus were found in 10 of the 20 samples from the gradient (Fig. 2A): LV (1/5), LC (5/5) and NA (4/5), but the species was absent in all samples of site AC (situated at 1,772–1,800 masl).

On average, the abundance of P. corethrurus accounted for 73% of the total earthworm density throughout the samples where the species was present. This percentage varied between sites LV, LC and NA at 92, 79 and 47%, respectively (Fig. 2B). In the sites where the species occurred, its average density was 273.5 ind. m−2, ranging from 16 to 704 ind. m−2 (Fig. 2C).

Pontoscolex corethrurus coexisted with exotic (two and four of the five samples in LC and NA, respectively) and native (1/5 in LV) species (Table 5). In contrast, P. corethrurus was found alone in three of the five monoliths of site LC, while only native species were found alone in site LV.

Table 5:

Earthworm community composition in each of the five monoliths at the four sites along an altitudinal gradient in central Veracruz, Mexico.

Discussion

Earthworm communities are determined by hierarchical organized factors: temperature operates at the highest hierarchical level, followed by soil nutrient and seasonality factors (Gerard, 1967; Fragoso & Lavelle, 1992; Briones et al., 2009; Eisenhauer et al., 2014). Compared to other types of terrestrial ecosystems, grasslands (which are the best carbon storage systems) are structurally simple and appear to be relatively homogeneous in terms of richness and functional complexity, particularly belowground (Stockdill, 1966; Stanton, 1988; Brown et al., 2004). Here, we found that along an altitudinal gradient, site (climate) can act as a barrier to distribution of peregrine earthworms and their abundance was determined significantly by soil fertility and grass quality.

Earthworm community

Along the altitudinal gradient studied here, 10 species (seven exotic and three native morphospecies) were recorded in the grassland. The exotic species are among the 51 exotic species recorded in Mexico, and the three morphospecies are among the 40 native species that are already known but still undescribed (Fragoso & Rojas, 2014). Of the ten species that we found, five are Neotropical (P. corethrurus, Onychochaeta windelei and three morphspecies), three are Western Palearctic (Lumbricus rubellus, Aporrectodea trapezoides and Octolasion tyrtaeum), one is Nearctic (Bimastos parvus) and one more is Eastern Palearctic (Amynthas gracilis). The earthworm diversity was similar (five species) between the tropical (LV and LC) and temperate sites (NA and AC), similar to the diversity (4–14 species) that has been observed in tropical and temperate forests (Fragoso & Lavelle, 1992).

The current state of knowledge allows little generalization about the distribution patterns of invasive earthworms, as is the case of P. corethrurus (Hendrix et al., 2008). However, Beddard (1912) suggested that: (a) temperate species tend to invade temperate regions and montane areas of the tropical regions, and (b) tropical earthworms only tend to invade tropical regions; that is, low temperatures limit their colonization of temperate areas. Our results show a trend for the influence of climate (site) on the distribution of earthworm species throughout the altitudinal gradient: the Palearctic and Nearctic species where only found in the temperate sites (NA and AC) and the Neotropical species only in the tropical sites (LV and LC). However, P. corethrurus and A. gracilis was found in one temperate (NA), in one tropical (LC) site, respectively. Some characteristics of native species (morph 1 and 3) and their soil habitats (e.g., high content of expending clay) might be resistant to introduction of P. corethrurus in the site LV, that is, because their presence around this site was registered 35 years ago (Lavelle, Maury & Serrano, 1981). Also just as recorded by Juárez-Ramón & Fragoso (2014) and this study (site AC) P. corethrurus does not coexist with L. rubellus and A. trapezoides. In contrast, P. corethrurus coexists with Palearctic (A. gracilis and O. tyrtaeum) species as observed by Huerta, Gaspar-Genico & Jarquin-Sanchez (2014) and Juárez-Ramón & Fragoso (2014), respectively. Furthermore, P. corethrurus coexist with Neotropical (morph 2 and O. windelei) species.

Rapid adaptations or mutations in known invasive species should be considered as likely mechanisms that could facilitate their spread into new habitats (Hendrix et al., 2008). Genetic studies have shown a high level of genetic diversity in populations of P. corethrurus and they are probably differentiated into cryptic species (Dupont et al., 2012; Cunha et al., 2014). Our findings suggest that P. corethrurus inhabiting temperate grasslands is a lineage different to sites LV and LC. The correct molecular identification of P. corethrurus is needed to comprehend their history of colonization and as a baseline for biology, ecology and ecotoxicology research on this species (King, Tibble & Symondson, 2008; Dupont et al., 2012; Cunha et al., 2014).

Conclusions

Our results showed that soil quality significantly determined the abundance of the earthworm community along an altitudinal gradient. In addition, climate was shown to be a barrier to distribution of peregrine earthworms as suggested by Beddard (1912). P. corethrurus inhabiting tropical and temperate grasslands probably have 2–3 different lineages or ecotypes. Further studies will be needed to elucidate the genetic diversity of P. corethrurus.

Supplemental Information

Raw Data Invasion P. corethrurus

Data (PCA and Linear Mixed Models)

Acknowledgements

We thank the farmers in Laguna Verde, La Concepción, Naolinco and Acatlán for allowing access to their properties. Special thank to Carlos Fragoso for support in the identification of earthworms. We are grateful to Rogelio Lara González for technical assistance. We thank Martha Novo and Rosa Fernández for help throughout the study. We also thank Diana Pérez-Staples and two anonymous reviewers for valuable comments and careful revision of the manuscript.

Additional Information and Declarations

Competing Interests

The authors declare there are no competing interests.

Author Contributions

Diana Ortiz-Gamino and Angel I. Ortiz-Ceballos conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Data Availability

Funding

Consejo Nacional de Ciencia y Tecnología (CONACyT) Mexico awarded a PhD scholarship (No. 251818) to Diana Ortiz-Gamino. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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